المؤلفون: Chavan Siddhi, Khatal Anil, Phalake Satish, Tetali Sujata, Patil Ravindra

المصدر: Journal of Horticultural Research, Vol 32, Iss 1, Pp 33-42 (2024)

مصطلحات موضوعية: algorithms, expression stability, relative expression, quantitative rt-pcr, ubc17, rli, znf, Plant culture, SB1-1110

الوصف: In quantitative reverse transcription polymerase chain reaction (qRT-PCR), normalizing target gene expression using a reference gene is an indispensable step to control the variability of RNA extraction yield, RNA integrity, reverse transcription efficiency, and PCR amplification. In the present study, we identified candidate reference genes with stable expression during grapes’ flowering and berry development stages. Ten genes, including ACT, CYP5, RLI, TUB, UBC, UBC17, UBC60, UFD1, VAG, and ZNF with relatively stable expression, were selected based on RNAseq data generated earlier in grape hybrid ‘ARI 516’. The expression of these candidate genes was tested at different stages of flowering and grape berry development. Five different algorithms such as RefFinder, geNorm, NormFinder, BestKeeper, and the comparative ΔCq method were used to test the expression stability of candidate genes. A comprehensive ranking obtained by RefFinder showed that UBC17, RLI, and ZNF were the most stable reference genes during flower and berry development stages. UBC17, RLI, and ZNF were calibrators to normalize the expression of VvAGL11 as a target gene to validate the worthiness of identified reference genes. The result demonstrated that newly identified reference genes could be successfully used to normalize the expression of the target gene accurately. These reference genes will provide more choices for selecting appropriate reference genes to normalize gene expression in grapes.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2353-3978Test

الوصول الحر: https://doaj.org/article/afc77d64e53846fc9a50075635d045bcTest

المؤلفون: Yang Li, Juan Luo, Fuyou Zhang, Jiajing Shang, Chunran Deng, Yingjie Feng, Ge Meng, Wenming Jiang, Xiaohui Yu, Guanhui Liu, Hualei Liu

المصدر: Frontiers in Microbiology, Vol 15 (2024)

مصطلحات موضوعية: DAstV-3, DAstV-4, GoAstV-1, GoAstV-2, quadruple fluorescence quantitative RT-PCR, Microbiology, QR1-502

الوصف: Avian astrovirus can infect a variety of poultry species and cause viral diarrhea, with a wide epidemic range strong pathogenicity and a high incidence. Among them, Duck astrovirus 3(DAstV-3), Duck astrovirus 4(DAstV-4), Goose astrovirus 1(GoAstV-1) and Goose astrovirus 2(GoAstV-2) are four types of astroviruses newly discovered in waterfowl in recent years. In order to realize the rapid detection of these four kinds of waterfowl stellate viruses, specific primers and probes were engineered to target a highly conserved region of ORF1b gene of DAstV-3, GoAstV-1 and GoAstV-2 and the ORF2 gene of DAstV-4, and a quadruple fluorescence quantitative RT-PCR method was developed. The results indicate that the method established in this study has good specificity and no cross reactivity with other pathogens. This method can detect viruses with a minimum concentration of 1 × 101 copies/μL for DAstV-4, GoAstV-1 and GoAstV-2, respectively, while the minimum concentration for DAstV-3 is 1 × 102 copies/μL. Compared with the routinely used RT-PCR method, the limit of detection by the multiplex RT-PCR lower. Both intra- and inter-assay variability tests revealed excellent reproducibility. This method was then used to analyze 269 field samples, and the results were verified by genome sequencing. In conclusion, this study presents a sensitive, accurate, and specific method for detecting DAstV-3, DAstV-4, GoAstV-1, and GoAstV-2 in a single reaction, enabling the monitoring and differential diagnosis of these four types of waterfowl astroviruses.

وصف الملف: electronic resource

العلاقة: https://www.frontiersin.org/articles/10.3389/fmicb.2024.1328243/fullTest; https://doaj.org/toc/1664-302XTest

الوصول الحر: https://doaj.org/article/2f7c6f795e6b408b994b9afe171dc0ebTest

المؤلفون: Yusuf A. Elgharbawy, Saad S. Ali, Ehab M. El-Nahas, Mervat M. Ali

المصدر: Journal of Advanced Veterinary Research, Vol 14, Iss 5 (2024)

مصطلحات موضوعية: Turkey Pox Virus, Quantitative RT-PCR, Veterinary medicine, SF600-1100

الوصف: Turkey Pox Virus (TKPV) is one of avipox virus affecting poultry breeding causing many economic losses due to general skin lesion on the non-feathered area of the body (cutaneous form) and may be fatal in case of diphtheritic form. Turkey breeding in Egypt is getting a lot of attention lately so the disease of turkey get more concern, the most important disease that causes loses is (TKPV). The most effective way to control the disease is vaccination the birds with suitable vaccine protecting against the circulating virus therefor this study for production of TKPV vaccine. TKPV was isolated by inoculation of eleven day old embryonated chicken egg on chorioallantoic membrane (CAM) then the egg adapted TKPV was propagated on chicken embryo fibroblast (CEF) till adaptation at the 15th passage when titer was reaching to log 10 5.5 TCID 50/ ml . The adapted virus was transmitted to Vero cell line to produce the qualified vaccine. All quality control measures approved that the vaccine is ready to be used to control the TKPV disease in Egypt. C4L like gene expression was employed to evaluate TKPV virulence via Quantitative RT-PCR to ensure TKPV attenuation that begin from the 15th passage till complete attenuation at the 20th passage.

وصف الملف: electronic resource

العلاقة: https://www.advetresearch.com/index.php/AVR/article/view/1719Test; https://doaj.org/toc/2090-6269Test; https://doaj.org/toc/2090-6277Test

الوصول الحر: https://doaj.org/article/bc0e190dceac48668b3f5c35c4fb30d3Test

المؤلفون: Jianxin Mao, Jiqi Li, Yan Wang, Zhihong Zhang

المصدر: Fruit Research, Vol 4, Iss 1, Pp 1-7 (2024)

مصطلحات موضوعية: quantitative rt-pcr, reference genes, non-climacteric fruits, strawberry, Plant culture, SB1-1110

الوصف: The rapid, reliable, and efficient characteristics of quantitative reverse transcription polymerase chain reaction (qRT-PCR) make it a highly advantageous method for assessing gene expression levels. The identification of stable reference genes is crucial for successful gene expression studies. Cultivated strawberry fruit has been extensively investigated as a model for studying the non-climacteric fruit ripening process. However, more research needs to be conducted on identifying suitable reference genes at different developmental stages of strawberry fruit. We selected the 'Yanli' and 'Chuliandeweidao' cultivars to screen potential reference genes in various tissues, organs, and developmental stages of strawberry fruit. Based on the analysis of high-quality haplotype-resolved genome and transcriptomic FPKM data, FaADPrf1 (ADP-ribosylation factor 1), FaGAPC2 (Glyceraldehyde-3-phosphate dehydrogenase), FaPPC1 (Peptidyl-prolyl cis-trans isomerase 1), and FaEF1-α (Elongation factor 1-alpha) were selected as candidate reference genes, along with the commonly used Fa26S rRNA, for normalization purposes. A qRT-PCR analysis showed 89.21% to 101.51% amplification efficiency for five candidate reference genes, with correlation coefficients (R2) exceeding 0.99. Reference genes' expression stability was assessed using GeNorm, NormFinder, BestKeeper, and Comparative delta-Ct method. RefFinder analysis determined that FaGAPC2 and FaADPrf1 were the most suitable reference genes, considering the results obtained from the abovementioned four methods. The calculated results were validated by studying the expression of FaMYB10, FaUGT1, and FaCHS in different developmental stages of 'Yanli' fruit. This validation confirmed that both FaGAPC2 and the combination of FaGAPC2 and FaADPrf1 could serve as suitable reference genes, with the combination of FaGAPC2 and FaADPrf1 being more suitable than the single FaGAPC2 in certain cases. In summary, we obtained suitable reference genes for research on cultivated strawberry fruit development, which will benefit further study on the ripening of non-climacteric fruits.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2769-4615Test

الوصول الحر: https://doaj.org/article/73ac0baa39384a7ba3eef19547a1e175Test

المؤلفون: Elgharbawy, Yusuf A., Ali , Saad S., El-Nahas , Ehab M., Ali , Mervat M.

المصدر: Journal of Advanced Veterinary Research; Vol. 14 No. 5 (2024): Special Issue (Veterinary Medicine and Food Safety Challenges); 785-788 ; 2090-6277 ; 2090-6269

مصطلحات موضوعية: Turkey Pox Virus, Quantitative RT-PCR

الوصف: Turkey Pox Virus (TKPV) is one of avipox virus affecting poultry breeding causing many economic losses due to general skin lesion on the non-feathered area of the body (cutaneous form) and may be fatal in case of diphtheritic form. Turkey breeding in Egypt is getting a lot of attention lately so the disease of turkey get more concern, the most important disease that causes loses is (TKPV). The most effective way to control the disease is vaccination the birds with suitable vaccine protecting against the circulating virus therefor this study for production of TKPV vaccine. TKPV was isolated by inoculation of eleven day old embryonated chicken egg on chorioallantoic membrane (CAM) then the egg adapted TKPV was propagated on chicken embryo fibroblast (CEF) till adaptation at the 15th passage when titer was reaching to log 10 5.5 TCID 50/ ml . The adapted virus was transmitted to Vero cell line to produce the qualified vaccine. All quality control measures approved that the vaccine is ready to be used to control the TKPV disease in Egypt. C4L like gene expression was employed to evaluate TKPV virulence via Quantitative RT-PCR to ensure TKPV attenuation that begin from the 15th passage till complete attenuation at the 20th passage.

وصف الملف: application/pdf

العلاقة: https://www.advetresearch.com/index.php/AVR/article/view/1719/1230Test; https://www.advetresearch.com/index.php/AVR/article/view/1719Test

الإتاحة: https://www.advetresearch.com/index.php/AVR/article/view/1719Test

المؤلفون: D’Amico-Willman, Katherine M, Sideli, Gina M, Allen, Brian J, Anderson, Elizabeth S, Gradziel, Thomas M, Fresnedo-Ramírez, Jonathan

مصطلحات موضوعية: Agricultural, Veterinary and Food Sciences, Biological Sciences, Genetics, Human Genome, Aetiology, 2.1 Biological and endogenous factors, Good Health and Well Being, DNA methylation, non-infectious bud failure, differentially methylated regions, Prunus interspecific hybrids, quantitative RT-PCR, methylome, Plant Biology, Crop and pasture production, Plant biology

الوصف: Almond [Prunus dulcis (Mill.) D.A. Webb] is an economically important nut crop susceptible to the genetic disorder, Non-infectious Bud Failure (NBF). Despite the severity of exhibition in several prominent almond cultivars, no causal mechanism has been identified underlying NBF development. The disorder is hypothesized to be associated with differential DNA methylation patterns based on patterns of inheritance (i.e., via sexual reproduction and clonal propagation) and previous work profiling methylation in affected trees. Peach (Prunus persica L. Batsch) is a closely related species that readily hybridizes with almond; however, peach is not known to exhibit NBF. A cross between an NBF-exhibiting 'Carmel' cultivar and early flowering peach ('40A17') produced an F1 where ∼50% of progeny showed signs of NBF, including canopy die-back, erratic branching patterns (known as "crazy-top"), and rough bark. In this study, whole-genome DNA methylation profiles were generated for three F1 progenies exhibiting NBF and three progenies considered NBF-free. Subsequent alignment to both the almond and peach reference genomes showed an increase in genome-wide methylation levels in NBF hybrids in CG and CHG contexts compared to no-NBF hybrids when aligned to the almond genome but no difference in methylation levels when aligned to the peach genome. Significantly differentially methylated regions (DMRs) were identified by comparing methylation levels across the genome between NBF- and no-NBF hybrids in each methylation context. In total, 115,635 DMRs were identified based on alignment to the almond reference genome, and 126,800 DMRs were identified based on alignment to the peach reference genome. Nearby genes were identified as associated with the 39 most significant DMRs occurring either in the almond or peach alignments alone or occurring in both the almond and peach alignments. These DMR-associated genes include several uncharacterized proteins and transposable elements. Quantitative PCR was also performed to analyze the gene expression patterns of these identified gene targets to determine patterns of differential expression associated with differential DNA methylation. These DMR-associated genes, particularly those showing corresponding patterns of differential gene expression, represent key targets for almond breeding for future cultivars and mitigating the effects of NBF-exhibition in currently affected cultivars.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/5r86s1gcTest

المؤلفون: Shengting Zhang, Xiuling Hu, Chunting Zhang, Yani Ju, Xin Liu, Yunlin Wei

المصدر: Frontiers in Microbiology, Vol 14 (2023)

مصطلحات موضوعية: anti-infection, anti-phage activity, bacteriophage, catecholamine, dopamine, quantitative RT-PCR, Microbiology, QR1-502

الوصف: Antiviral drug development is important for human health, and the emergence of novel COVID-19 variants has seriously affected human lives and safety. A bacteriophage—a bacterial virus with a small and simple structure—is an ideal experimental candidate for studying the interactions between viruses and their hosts. In this study, the effects and mechanisms of catecholamines on phages were explored, and dopamine (DA) was found to have general and efficient anti-infection effects. A clear dose-dependent effect was observed when different phages were treated with DA, with higher DA concentrations exhibiting stronger anti-phage activity. The half maximal inhibitory concentration values of DA for vB-EcoS-IME167, T4 Phage, and VMY22 were determined as 0.26, 0.12, and 0.73 mg mL−1, respectively. The anti-phage effect of DA increased with treatment duration. In addition, the anti-infection activities of DA against vB-EcoS-IME167, T4 Phage, and VMY22 were increased by 105, 104, and 104 folds compared to that of the control. This ability of DA was observed only in phages and not in the host bacteria. Morphological changes of phages were observed under transmission electron microscopy following their treatment with DA, and considerable changes in adsorption were confirmed via quantitative reverse transcription polymerase chain reaction. These results suggest that the anti-phage effect of DA is primarily due to the destruction of the external structure of the phage. This study, to the best of our knowledge, is the first to report the universal anti-phage infection effect of dopamine, which provides novel information regarding DA and forms a basis for further research and development of antiviral drugs. Moreover, it provides a new perspective for the research about the defense and counter-defense of bacteria and bacteriophages.

وصف الملف: electronic resource

العلاقة: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1272447/fullTest; https://doaj.org/toc/1664-302XTest

الوصول الحر: https://doaj.org/article/7a134fd319374dd09a0a50eb42d5b31aTest

المؤلفون: Borah, Sudarshana¹, Saikia, Banashree, Bora, Dipsikha¹, Bhorali, Priyadarshini¹

المصدر: Indian Journal of Genetics and Plant Breeding (The) 82(2):217-223. 2022

المؤلفون: Asami Okada, Misuzu Yamada‐Yamashita, Yukari Tominaga, Kyoka Jo, Hiroyasu Mori, Reiko Suzuki, Masashi Ishizu, Motoyuki Tamaki, Yuko Akehi, Yuichi Takashi, Daisuke Koga, Eisuke Shimokita, Fuminori Tanihara, Kiyoe Kurahashi, Sumiko Yoshida, Yukari Mitsui, Shiho Masuda, Itsuro Endo, Ken‐ichi Aihara, Shoji Kagami, Masahiro Abe, Kevin Ferreri, Yoshio Fujitani, Munehide Matsuhisa, Akio Kuroda

المصدر: Journal of Diabetes Investigation, Vol 13, Iss 7, Pp 1140-1148 (2022)

مصطلحات موضوعية: DNA methylation, Quantitative RT‐PCR, Type 1 diabetes, Diseases of the endocrine glands. Clinical endocrinology, RC648-665

الوصف: Abstract Aims/Introduction Several research groups have reported methods for quantifying pancreatic beta cell (β‐cell) injury by measuring β‐cell‐specific CpG unmethylation of the insulin gene in circulation using digital droplet PCR or next‐generation sequencing. However, these methods have certain disadvantages, such as the need to consider the background signal owing to the small number of target CpG sites and the need for unique equipment. Materials and Methods We established a novel method for detecting four CpG unmethylations of the insulin gene using two‐step amplification refractory mutation system PCR. We applied it to type 1 diabetes (T1D) patients with a wide range of disease durations and to healthy adults. Results The assay showed high linearity and could detect a single copy of unmethylated insulin DNA in experiments using methylated and unmethylated plasmid DNA. The unmethylated insulin DNA level in the type 1 diabetes group, whose β‐cell mass was considerably reduced, was similar to that of healthy adults. An inverse correlation was observed between copy number and disease duration in patients with unmethylated insulin DNA‐positive type 1 diabetes. Conclusions We developed a novel method for detecting unmethylated insulin DNA in circulation that can be performed using a conventional real‐time PCR system. This method would be useful for analyzing dynamic profiles of β‐cells in human disease such as type 1 diabetes.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2040-1116Test; https://doaj.org/toc/2040-1124Test

الوصول الحر: https://doaj.org/article/885cf638d27f4b7d8643cfc191e2e4cfTest

المؤلفون: Marika Nakagawa, Keiko Okano, Yuya Saratani, Yosuke Shoji, Toshiyuki Okano

المصدر: Zoological Letters, Vol 8, Iss 1, Pp 1-17 (2022)

مصطلحات موضوعية: Cryptochrome, Circadian clock, Photoperiodicity, Sun compass, Quantitative RT-PCR, Eye, Zoology, QL1-991

الوصف: Abstract Photoperiodic responses are observed in many organisms living in the temperate zones. The circadian clock is involved in photoperiodic time measurement; however, the underlying molecular mechanism for detection of the day length remains unknown. We previously compared the expression profiles of the Cryptochrome(Cry) genes in the zebrafish eye and reported that Cry1ab has a double peak with variable expression duration depending on the photoperiod. In this study, to understand commonalities and differences in the photoperiodic responses of ocular Cry genes, we identified Cryptochrome genes in two other teleost species, goldfish and medaka, living in temperate zones, and measured ocular Cry mRNA levels in all of the three species, under different photoperiods (long-day [14 h light: 10 h dark] and short-day [10 h light: 14 h dark] and in constant darkness. Cry1ab mRNA levels did not show dual peaks in goldfish or medaka under the examined conditions; however, the mRNA expression profiles of many Crys were altered in all three species, depending on the day length and light condition. Based on their expression profiles, Cry mRNA peaks were classified into three groups that better synchronize to sunrise (light-on), midnight/midday (middle points of the dark/light periods), or sunset (light-off). These results suggest the presence of multiple oscillators that oscillate independently or a complex oscillator in which Cry expression cycles change in a photoperiod-dependent manner in the eye.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2056-306XTest

الوصول الحر: https://doaj.org/article/2a9d47c4c3ae44b7840959c28e42d245Test